Several examples have been reported of cross-linking between complementary oligonucleotides. The purpose of such cross-linking often has been to disrupt the function of a nucleic acid, primarily by cleavage of the target. It is known to cross-link nucleic acids in a positionally uncontrolled manner with UV exposure. However, Grineva, and Karpova, FEBS., 1973, 32, 351-355, appear to have been the first to covalently cross-link complementary strands of oligonucleotides at a specific site utilizing controlled chemistry. A nitrogen mustard was attached to the 3' terminal ribose unit of an oligonucleotide via an acetal linkage or to the 5' end of an oligonucleotide via a phosphoramide linkage. On hybridization, the reactive mustards covalently cross-linked to the complementary strand via alkylation of the ternary heteroaromatic nitrogen atom at the 7-position of guanine or adenine. However, the alkylated base thus formed is a quaternary charged species that is subject to rapid chemical degradation via imidazole ring opening followed by cleavage of the targeted strand.
Enzymes are known to remove alkylated bases from nucleic acids. Indeed, such enzymatic removal is implicated in DNA repair mechanisms. The destruction of a targeted alkylated nucleic acid theoretically could be utilized for antimicrobial or antitumor therapy. However, cross-linking alkylation reactions and subsequent degradations are not desirable where host cell viability is necessary.
Summerton and Bartlett have proposed another type of alkylated oligonucleotide wherein an .alpha.-bromomethylketone attached to the 4-position of a cytidine nucleotide spans the major groove and alkylates the 7-position of a complementary guanine residue in a targeted strand. [See, J. Mol. Biol., 1978, 122, 145-162; J. Theor. Biology, 1979, 78, 61-75; and U.S. Pat. No. 4,123,610]. As with the nitrogen mustards noted above, this alkylation yields a charged species that effects cleavage of the targeted nucleic acid strand following alkylation.
A further example of a covalent cross-linkage to the 7-position nitrogen of guanine is described by Meyer et. al., J. Am. Chem. Soc., 1989, 111, 8517. The authors attached an iodoacetamidopropyl moiety to the 5-position of a thymidine nucleotide of DNA. The iodoacetamidopropyl moiety subsequently alkylated the 7-position of a guanine nucleotide at a position two base pairs down the complementary strand. Cleavage of the targeted strand was observed at various times and temperatures.
Matteucci and Webb have described a hybridization triggered cross-linking process where an N6,N6-ethano-adeninc or N4,N4-ethanocytosine alkylates an appropriately positioned nucleophile in a complementary strand. [See, Nucleic Acids Res., 1986, 14, 7661; Tetrahedron Letters, 1987, 28, 2469-2472.] This process has been designed to inactivate the normal function of the targeted DNA either by forming a stable adduct or by hydrolytic or enzymatic cleavage.
A cross-linkage similar to the above-noted N6,N6-ethano linkage was described by Ferentz, et al., J. Am. Chem. Soc., 1991, 113, 4000, who utilized either an ethyl disulfide or a propyl disulfide linkage to connect the N6-positions of adeninc residues on a self-complementary oligonucleotide.
Lee, et al., Biochemistry, 1988, 27, 3197-3203, described the interaction of psoralen-derivatized methyl phosphonate oligonucleotides with single-stranded DNA. According to this method, irradiation effects cross-linking which, in turn, inactivates DNA. Psoralen cross-linkages occur only between pyrimidine bases to join single-stranded or, more commonly, double-stranded structures.
The generation of an abasic site on a nucleic acid has been implicated in the above-noted enzymatic removal of alkylated bases from nucleic acids. Manoharan, et al., J. Am. Chem. Soc., 1987, 109, 7217 and Manoharan, et al., J. Am. Chem. Soc., 1988, 110, 2690, have characterized abasic sites in oligonucleotides utilizing .sup.13 C NMR spectroscopy. Abasic sites also have been created on nucleic acids to attach the intercalator 9-aminoellipticine to nucleic acids. Vasseur, et al., Nucleosides & Nucleotides, 1989, 8, 863-866, reported that the trimer Tp(Ap)Pt, where (Ap) is an apurinic site yielded a mixture of products when reacted with the intercalator 9-aminoellipticine. The reaction produced a desired Schiff's base adduct between the apurinic site and the 9-aminoellipticine, but also resulted in cleavage of the phosphate backbone. This work was extended by Bertrand, et al., Nucleic Acids Research, 1989, 17, 10307, to the interaction of 9-aminoellipticine and a 13-mer oligonucleotide. The 13-mer oligonucleotide contained only a single purine nucleotide. Acid treatment resulted in loss of the purine base to yield an unstable abasic site. The 9-aminoellipticine was successfully linked to the abasic site in the oligonucleotide by reducing the Schiff's base adduct during the reaction. Using an oligonucleotide having a single purine site, the authors were able to study the coupling of the intercalator 9-aminoellipticine to the oligonucleotide. It is unlikely, however, that oligonucleotides which bind to sequences of biological significance contain only a single purinic site.
Other abasic site-containing oligonucleotides have been assembled by synthetically incorporating abasic nucleoside precursors into an oligonucleotide. For example, Philippe, et al., Tetrahedron Letters, 1990, 31, 6347-6350, incorporated a 2-deoxy-D-ribose having a primary amide function attached via a pentamethylene chain to its 1'-position. 1,2-Dideoxy-D-ribofuranose and butane 1,3-diol likewise have been incorporated into oligonucleotides. [See, P. lyer, et al., Nucleic Acids Research, 1990, 18, 2855. ]
Groehke, et al., Helvetica Chimica Acta, 1990, 73, 608, reported the preparation of a (tert-butyl)dimethylsilyl-protected deoxy-D-ribose and its incorporation into oligonucleotides utilizing solid state techniques. Reoc'h et al., Tetrahedron Letters, 1991, 32, 207, used 1-(o-nitrobenzyl)-2-deoxy-D-ribofuranose in the synthesis of other oligonucleotides. None of these chemically generated abasic sites have been utilized to effect cross-linking.
Application PCT/US91/01822, filed Mar. 19, 1991 and entitled Reagents And Methods For Modulating Gene Expression Through RNA Mimicry, discloses effective use of oligonucleotides that mimic RNA structure. The entire disclosure Application PCT/US91/01822, which is assigned to the assignee of this application, is herein incorporated by reference. The oligonucleotides disclosed in PCT/US91/01822 are selected to mimic a portion of an RNA encoded by a gene. The RNA-mimicking oligonucleotides interfere with or modulate gene expression by interfering with protein-RNA interactions.
In order to mimic an RNA, an oligonucleotide must assume and retain the RNA's secondary and tertiary structure. Although the physical and chemical forces that normally retain the RNA in its secondary and tertiary structure are non-covalent in nature, one way to fix an RNA mimic in the necessary secondary and tertiary structure would be to covalently cross-link the mimic. However, presently-known techniques for cross-linking nucleic acid strands either lead to strand cleavage, disruption of structure or Watson/Crick hydrogen bonding, or are only useful for limited, specific sequences or at specific locations in a sequence.
Cross-linkages between two oligonucleotide strands or between regions of a single strand would be undesirable where the cross-linkages could destroy the strands or strand or could unduly modify the conformational structure of the oligonucleotide. Cross-linkages also would be undesirable where they do not allow the cross-linked product to approximate the conformation of natural nucleic acids. Heretofore, there has be no suggestions in the art of cross-linkages or methods of cross-linking that do not destroy the strands, that allow suitable conformations, that are useful on various sequences and at various positions within the sequences, and that allow normal ranges of features such as the "tilt" and "propeller twist" features found in naturally occurring nucleic acid duplexes. Accordingly there remains a long-felt need for nucleic acid cross-linkages and methods of cross-linking nucleic acids.